Method and device for 3 dimensional imaging of suspended micro-objects providing high-resolution microscopy

ABSTRACT

A method for high-resolution image recording of at least one object with a microscope, includes the steps of: (a) positioning the object in a receptacle being arranged in the optical axis of the microscope, (b) generating at least two first data sets per object which represent intermediate images of the object with at least two different orientations relative to the optical axis of the microscope, wherein the different orientations of the object are provided by moving the object relative to the receptacle, and (c) evaluating the data sets for obtaining quantitative three dimensional information.

The invention is related to methods and devices for high-resolutionimage recording of at least one object, in particular with a microscope.

1) THE BACKGROUND OF THE INVENTION

A) Microscopy—in Biological Research.

The development of fluorescent bio-molecular probes—especiallyfluorescent proteins enabling the observation of sub-cellular processand structure inside living cells, has come to pass as a renaissance inlight microscopy. Now a variety of evolving bio-chemical techniquesincorporate and/or are based upon combining fluorescent molecular probesand light microscopy. They provide both qualitative and quantitativevisualization of specific molecular dynamics underlying live cellularactivities. However, inasmuch as it is now clear that these biologicalprocesses depend upon spatio-temporal compartmentalization, the majorfocus for development in light microscopy, is to overcome certainsystematic problems that obstruct our ability to render simple, yetquantitatively accurate three-dimensional mapping of fluorescent signalsinside living cells (viz. 3-D microscopy).

Three Major Systematic Problems in 3-D Microscopy

The ability to render three-dimensional maps of fluorescent signals frommicro objects visualized by light microscopy is limited mainly by threesystematic artifacts.

a) Axial Aberration—the Elongation Effect

A central issue in light microscopy arises from limitations due to thespatial resolution of images. This is dependent upon the objective lensused and the geometry of the light focused by the lens. In general,microscope objectives with a high magnification and a high numericalaperture are used for achieving the best resolution (e.g. 63×; 100×/N.A.1.4 oil immersion). However, there exist physical limitations for lightcollection through the glass lens of any objective. In particular, thexy resolution is always (at least two times) greater than in the z-axis(a.k.a “optical” axis). Specifically, x,y plane resolution is around100-150 nm, whereas the resolution along the z-axis is much lower(around 300 to 500 nm), and this fact results in a major systematicartifact of light microscopy—i.e. “axial aberration”, whereby aspherical object at the focus of a microscope objective in fact appearsto be elliptic in shape, with its largest extension along the z-axis,i.e. the light path. A schematic representation of this type of opticalaberration (the “elongation” effect) is shown in FIG. 9. The axes of themicroscope optics are x (9.1), y (9.2) and z (9.3). A feature 9.4 whichis originally circular in reality appears to be elongated 9.5 due tooptical aberration, and this problem is one of the major obstacles toovercome in 3 dimensional imaging microscopy.

b) Chromatic Aberration—Axial Misalignment of Multiple Wavelengths.

In addition to the problems of optical aberration and diminished axialresolution that reduce the ability to visualize in 3-D on a lightmicroscope, another problem is referred to as “chromatic aberration” or“axial chromatic aberration”. Chromatic aberration occurs inapplications where multi color images are acquired (Beyer). Whenperforming z-scans at different wavelengths the light diffraction at theglass-to-medium-interface and within the whole microscope set-up dependsupon the wavelength. As a consequence the focus for differentwavelengths varies as a function of the z-axis displacement. Varioustypes of correction are used to overcome chromatic aberration, forexample, in confocal-microscopy (see below) a calibration of the “zero”position (unique to just one z-axis position) for all colors can beperformed by an alignment of the light paths for the differentwavelengths using multi colored (artificial) spatial-calibration samples(so-called “Focal Check Microparticles”). However, inasmuch as thecalibration is unique to just one z-axis focal position, if a shift infocus occurs a systematic misalignment will again persist.

c) Out of Focus Light, and Diffraction Effects.

The third problem of three-dimensional fluorescence microscopy is thatthe micro-object itself comprises a complex three-dimensional form, andas such interferes with image visualization from the focal plane. Thisfact gives rise to a host of related problems for which there is nosingle general solution. Nonetheless, these problems are inextricablylinked and must be considered critically in order to achieve truethree-dimensional rendering. In general, these problems stem from lightdistortion caused by both the object itself, and the non-linearcharacteristics of light diffusion between different focal planes. Thesesorts of problem become critical in fluorescence microscopy whereso-called “out-of-focus-light”, (light from parts of an object layingoutside the focal plane) contribute to what is observed at the focalplane. This light compromises the crispness of the image, inasmuch as itintroduces an out-of-focus “haze” into the image focal plane.

There are two ways of eliminating or reducing out-of-focus haze: i)confocal microscopy (see below), and ii) deconvolution. The latter is acalculation-intensive, algorithm-based mathematical method forsharpening images (from any source) that contain out-of-focus light. Ingeneral, the method requires that an axial stack of images be collectedfrom the sample, at small (e.g. 50-500 nm) steps. The axial stack maythen be processed using special algorithms that take into account avariety of optical parameters including the objective lens, andexcitation/emission wavelengths. The corrected stack of images is thenconverted into a three-dimensional model of the object by eitherremoving or reassigning the identified out-of-focus light (Egner,Markham). Using adequate graphics computing power, thisthree-dimensional model may then be rendered into animations allowingthe object to be observed from any arbitrary viewing point.

Disadvantages of this approach are that it is calculation intensive(which costs time and processing power), and that it suffersinaccuracies and artifacts due to its extensive dependence uponcalculation-based assumptions and/or corrections that must be applied atmultiple stages during the processing procedure. It must also be notedthat the best types of deconvolution algorithm rely upon priormeasurement of a so-called PSF (Point-Spread-Function) specific to anygiven optical configuration (ie the microscope set-up). Put simply, thePSF is a measure of light diffusion from a sub-resolution point within agiven focal plane, and can therefore be used to “re-map” out-of-focuslight back into its appropriate 3-D voxel. However, a major problem withapplied algorithms using PSF, is that it is extremely difficult tomeasure a “good” PSF. In particular, a major problem arises from thefact that the PSF in any given sample is itself altered as a function ofthe axial distance through the sample (Sedat). This fact results in anysingle PSF being representative only of a single focal plane, andtherefore distorts reconstructions based upon z-axis stacks whereclearly the z-axis is deflected in order to scan throughout the volumeof a given sample. Finally, in addition to out-of-focus light, thesample (as stated above) alters the diffusion of light through its ownvolume by diffraction. This gives rise to a further group of problems,whereby light emission from the focal plane is deteriorated due toshading or diffraction do light by optically dense regions within theobject itself (e.g. cell) that lay in between the microscope lens andthe fluorescent features being imaged.

Advanced Techniques for Improved 3-D Fluorescence Microscopy

Three-dimensional imaging of micro objects requires that the aboveproblems be addressed, and this may in part be achieved using numeroustypes of novel approach. Herein, a brief description summarises some ofthe advanced microscope techniques at the cutting edge of what iscurrently available. However, it should be noted that all will beimproved substantially by the utility of the invention described herein.On this point, as for conventional fluorescence microscopy, all thesetechniques (without exception) achieve 3-D rendering by mechanicallyscanning the focus through multiple z-axis acquisitions at small (nm)intervals, collected from the sample volume. As such the micro objectmust, therefore, be immobilized by adherence to an optically transparentsurface substrate (normally a 150 micron thick glass cover-slip). Theresulting image “z-stack” must then be treated by calculation-intensiveprocessing to yield 3-D rendering.

Single-photon excitation confocal fluorescence microscopy uses focusedlaser light for fluorescence excitation and, in general, a pinhole inthe path of fluorescence emission, which allows in focus light derivedfrom the x,y image plane to pass, but effectively rejects out-of-focuslight. Fluorescence light measured using pinhole systems is detectedusing photo-multipliers and a scanning device. By way of an alternative,some commercial confocal systems use a so-called “Spinning-Disc” (Nipkowdisk) system that achieves much the same result by rejectingout-of-focus light. However, the detection system differs inasmuch as itcomprises a CCD camera, affording greater speed of acquisition. Eitherway, the advantage of confocal microscopy is that out-of-focus haze isgreatly reduced, and by performing a z-scan, stacks of confocal imagescan be generated from a sample volume, in order to build athree-dimensional rendering of the imaged volume. Note that thisapproach still suffers from chromatic and axial aberration problems.

Multi-Photon Excitation Confocal Fluorescence Microscopy

A method for improving resolution in fluorescence microscopy is basedupon the use of multi photon laser excitation. Fluorescence excitationof a fluorophore occurs at a certain wavelength λ nominally determinedby its specific excitation absorption maxima. Efficient absorption of asingle photon at this wavelength results in excitation and emission offluorescent light (conventional fluorescence microscopy). However,excitation may also be achieved by simultaneous absorption of twophotons of lower energy, displaying wavelengths approximately half theexcitation maxima. This mode of so-called “multiphoton” excitation isconsidered to be “biphotonic or two-photon” induced fluorescence, and ismade possible by grace of high energy pulsed lasers. In general thismode of excitation can be considered a means to excite fluorescencefrom, for example, a blue-green absorbing fluorophore using multi-photonexcitation from a near-infra-red laser emitting sub-microsecond pulsesof light. Inasmuch as the two photons of near-IR light are aligned andcollide only at the focal plane of the optical set-up, the energydensity of this multi photon excitation is concentrated solely at asingle femtoliter volume within the microscope's focal plane. As such,multi-photon excitation is intrinsically confocal by nature. In effectthis approach gives a pure, and efficient image free from “out of focus”fluorescence. The disadvantage of multi photon fluorescence microscopyis the requirement for high energy pulsed lasers to be attached to themicroscope, resulting in high cost and large, difficult to manageequipment assemblage, maintenance and application.

4Pi Confocal (Theta) Microscopy, Standing-Wave Microscope (SWM),Incoherent Illumination Interference Image Interference Microscopy (I5M)

The generation of higher resolved three-dimensional images of cells canbe improved by a combination of the techniques mentioned above andmodifications of the optomechanic set-up. The use of two separateobjective lenses for excitation and collection of fluorescence emissionlight leads to a smaller detection volume element and an equilateralresolution some 4 times higher than for conventional fluorescencemicroscopy (Egner). This technique is used in combination with multiphoton fluorescence microscopy. In a 4Pi confocal fluorescencemicroscope two opposing microscope objective lenses are used toilluminate a fluorescent object from both sides and to collect thefluorescent emissions on both sides. Constructive interference of eitherthe illumination wave fronts in the common focus or the detection wavefronts in the common detector pinhole results in an axial resolutionapproximately four times higher than in a confocal fluorescencemicroscope (Hell). The excitation/observation volume can be considerablydecreased when the detection axis is rotated by an angle Theta (e.g.90°) relative to the illumination axis as in Theta Microscopy (Lindek).Both methods bring along substantial limitations for the sample carrierand the microscope objective, which can be used. In addition there is ahuge effort involved when aligning the two focal volumes of theobjective lenses, which has to be done with sub-micrometer precision.

B) Micro Electrode/Fluidics Chamber(s) for Three DimensionalManipulation of Micro Objects

Holding and lifting micro objects by negative dielectrophoresis in awell defined electric field minimum has been described since 1992 (Fuhr,G. et al. “Biochim. Biophys. Acta” 1108, 1992, 215-223). First, planartwo-dimensional arrangements of micro electrodes have been used. Theycontained for example four electrodes with a tip-to-tip distance of 100to 200 micrometers. Holding and lifting objects in these so-called“field traps” was only possible using alternating fields. Rotationalfields had only limited trapping efficiency and were very sensitive tohydrodynamic streaming (Schnelle, Th. et al. “J. Electrostatics” 46,1993, 13-28, Schnelle, Th. et al. “J. Electrostatics” 50, 2000, 17-29,Schnelle, Th. et al. “Appl. Phys. B” 70, 2000, 267-274, Reichle, Ch. etal. “Biochim. Biophys. Acta” 1459, 2000, 218-229). The development ofso-called CellProcessors—three-dimensional electrode arrangements led to“field cages” consisting of eight electrodes and building up closedelectric field cages (Schnelle, Th. et al., 1993, see above, Müller, Th.et al. “Biosensors & Bioelectronics” 14, 1999, 247-256, Reichle, Ch. etal. “Electrophoresis” 22/2, 2001, 272-282). “Cell Processors” containingdielectric field cages (DFCs) have been used in combination with avariety of high resolution optical techniques applied to micro objects,such as fluorescence correlation spectroscopy (FCS, Schnelle, Th. et al.“Electrophoresis” 21, 2000, 66-73), force measurements using lasertweezers (Fuhr et al. “Appl. Phys. A.” 67, 1998, 385-390),electro-rotation (Schnelle et al., see above), measurement ofligand-receptor binding forces (Reichle et al. 2001, see above) andconfocal laser scanning microscopy (Müller, Th. et al. “EuropeanBiophysics Journal” 29/4-5, 2000, 12D-3 (Poster); Wissel, H. et al.“American Journal of Physiology Lung Cell Mol. Physiol.” 281, 2001,L345-L360).

2) OBJECT OF THE INVENTION

The object of the invention is to provide improved high-resolutionmeasuring methods, in particular imaging methods avoiding thedisadvantages of conventional methods. It is a particular object of theinvention to provide a generally applicable solution that enhanceshigh-resolution 3-D imaging methods by overcoming, diminishing oravoiding completely the systematic artifacts of light based microscopydescribed above. The invention shall provide an altogether new approachfor the generation of three-dimensional image series describing bothquantitatively and qualitatively a more accurate spatial map of anygiven fluorescent, bioluminescent, or auto-fluorescent micro-object—e.g.a live mammalian cell labelled with fluorescent molecules. Anotherobject of the invention is to provide a device that may be implementedto any (and all) existing fluorescent microscope techniques (asdescribed above).

3) SUMMARY OF THE INVENTION

Generally, the high-resolution image recording according to theinvention comprises a positioning of at least one object in a receptaclein the optical axis of a microscope imaging system, generating at leasttwo intermediate images of the object with different orientations inspace and evaluating an object image from the intermediate images,wherein the different orientations of the object are provided by movingthe object as such relative to the receptacle. The feature of movingonly the object while the imaging system and the receptacle are kept atfixed positions (e.g. in a laboratory system) has the advantage ofproviding high-resolution images without troublesome changes of theopto-mechanical features of the imaging system. The term differentorientation refers generally to different geometrical arrangements ofthe object relative to the optical axis. The arrangements can beobtained by translations and/or rotations.

The invention can avoid completely (1 and 2), or minimize (3) thefollowing problems in 3-D light microscopy:

-   1. Axial aberration—Elongation effect of the imaged features.-   2. Chromatic aberration—axial misalignment at multiple wavelengths.-   3. Shading/Diffraction effects due to optically dense regions, and

The invention also optimizes completely (4 and 5), and enhances greatly(6) the following procedures of 3-D light microscopy:

-   4. 3-D object rendering—facile 3-D characterization, NO CALCULATION    NEEDED.-   5. PSF based algorithm remapping of “haze”—PSF required from single    voxel only.-   6. Optical Resolution—by using sampling from only one x,y plane to    detect axial shape distribution

One basic idea of the invention described herein is to use in particulara DFC in order to freely translate and/or rotate the micro object itselfin order to record different image planes and to keep the measurementvolume itself motionless (or to move it in just one dimension e.g.scanning).

In the context of the current invention these movements of the microobject—e.g. a live suspended biological cell—are preferably realized bytime dependent ac electric fields and negative dielectrophoretic forces.They are preferably generated in fluidic micro-chips containing DFC'sthat are customized to achieve specific movements of the micro object inthree dimensional space. In this context we have implemented andsubstantiated that a DFC comprising eight micro electrodes forming aclosed field cage, provides sufficient stability and control to achievethese movements. Ideally, the dimensions—diameter and distance betweenelectrodes—are preferentially within the range of the object's size,i.e. between 1 and 1000 μm. The electrodes were fixed within amicrofluidic chamber which was made of a transparent material suitablefor high resolution microscopy, e.g. glass with 150 μm thickness.Negative dielectrophoresis was induced by applying electric ac fields ina frequency range of 100 kHz to 100 MHz. Field cages were generated, forexample, by applying rotational fields to the two planes of fourelectrodes each with a phase shift of 90° between the electrodes withinone plane and of 180° between the planes. During micro object rotationthis mode yielded an efficient dielectric field cage and highly stablepositioning in x-, y- and z-directions.

Thus, the invention is based on this surprising and unexpected result ofthe inventors according to which micro-objects suspended in a fluid canbe manipulated with electric field forces with a precision sufficientfor an evaluation of the object image from the intermediate images. Theprecision of rotation even was obtained despite asymmetry and/orgravitation effects. Thus, it has been found that the object can bepositioned stably in x-, y- and z-directions. It can be rotated around adefined axis and oriented by addressing the electrodes with a suitedsignal characterized by time dependent amplitude, frequency and phase.

The physical reason for the rotation of a micro object is a polarizationof its charges/dipoles within a rotational field. The torque isdependent upon the field strength, the field frequency and the passiveelectric properties of the object in relation to the suspending medium.The torque can be deliberately induced by using appropriate rotationalfields, i.e. certain phase shifts between the electrodes and/or acertain geometry of the electrode arrangements. Computer/user controlledprotocols will be adapted that allow the electrodes to be alternatelytriggered in order to achieve fixed protocols of micro object movement,and facile directional and speed control. The resulting device shall becapable of allowing the user to select and change the rotational axis,speed of rotation, and extent/angle of deflection.

With this device rotating the object can be realized in a continuousfashion or for defined time periods and angles. For example, ahorizontal rotation can be changed to a vertical rotation or a “random”rotation while maintaining the same z-position. Small changes of therotational axis can be achieved by varying the amplitude and/or thephase at least at a single electrode. Moreover, a well-defined variationof the z-position can be achieved by varying the amplitude and/or thephase within one electrode plane, and rotational axes can be changedwithout affecting the positioning stability.

For caging objects within a DFC without inducing a torque AC fields canbe used. The dipole moment in z is then zero and forcing inz-direction—i.e. the positioning along the z-axis against the force ofgravity—is achieved merely by higher moments, e.g. quadruple moment.This works best for a ratio of object diameter compared to tip-to-tipdistance of the DFC electrodes of 1:4 to 1:10. Preferred fieldmodulations are adapted for changing a horizontal rotation to a verticalrotation or a “random” rotation while maintaining the same z-position,applying small changes of the rotational axis—typically 1° to 10°—byvarying the amplitude and/or the phase at least at one electrode, orvarying the z-position—typically 50 nm to 10 μm—by varying the amplitudeand/or the phase within one electrode plane. If the object to beinvestigated has an asymmetrical shape, the electrodes can be controlledsuch that the object is rotated relative to a fixed centre of rotation.A variation of angular velocity and/or centre of rotation can becompensated by a field amplitude modulation. In terms of a homogeneouscompensation, the electric field forces preferably are generated by aplurality of electrodes (about 8 or more) surrounding the object.

According to an alternative embodiment of the invention the micro objectmay be moved by holding the object at a fixed position by means ofelectric field forces and by rotating the object by means of opticalforces as generated e.g. with laser tweezers.

Image Series Generated from Micro Object Rotation Using the InventionContain Characteristic 3-D Spatial Information that is Free fromArtifacts.

Generally, the high-resolution image recording according to theinvention comprises a positioning of at least one object in a receptaclein the optical axis of a microscope imaging system, generating at leasttwo intermediate images of the object with different orientations inspace and evaluating an object image from the intermediate images,wherein the different orientations of the object are provided by movingthe object as such relative to the receptacle. Clearly, rotating a microobject while maintaining the imaging system, optics and samplereceptacle in a fixed position has the advantage of generating an x,yimage series that reveals implicitly 3-D details that are transientlyaxial to the focal plane, but without axial or chromatic aberrations.Moreover, this 3-D visualization is facile, inasmuch as it does notrequire troublesome axial changes in the focal plane that normallyrequire complex opto-mechanical devices.

Thus, in effect the invention allows, on-line, “real-time” 3-D movies tobe generated of micro-objects that should normally require “virtual”rendering by off-line, convoluted and expensive methodologies.

Utility and Further Advantages of the Invention:

The methods of the invention are implemented with an optical microscopeimaging system as e.g. a modified conventional fluorescence microscopebeing equipped with a two-dimensional camera or with other detectorelements being arranged point-wise, as a line or two-dimensionally. Inthe context of the present invention, “high-resolution imaging” isdefined as spatially mapping micro-objects with maximum opticalresolution (i.e. on the x,y plane of axes ˜100 nm) without recourse toz-axis deflection that should nominally introduce axial aberration and aloss of resolution. A micro-object to be imaged is a biological orsynthetic object with a typical cross-section in the range of 1-100 μm.Imaging an object generally comprises mapping of topographical objectfeatures. The object features comprise the complete object or only partsthereof, they are located on the surface, or in the volume of theobject. The 3-D object shape may be evaluated from the intermediateimages, and by grace of the invention specific rotations of thesuspended cell, around a fixed xyz coordinate gives optimal opticalaccess to every location within the cell. Thus, the invention makes itpossible to generate image planes from different angles and objectplanes without moving the microscope objective or the scanning table ofthe microscope. For the application of deconvolution algorithms usingPSF based calibrations, this has the added advantage that only a singlePSF (measured a fixed distance from the objective and coincident withthe focal plane) needs to be applied.

Continuous imaging of controlled changes in position of the object byrotating it around at least one horizontal axis and one vertical axis isvery useful for a reduction of the calculation effort required for athree dimensional imaging. A film series (see figures) of cell rotationimaged with a camera at relatively high speed (1-50 images/second)results in a “real” recording of three-dimensional structure, therebyreplacing the virtual 3-D rendering generated from conventionaloptomechanical z-stacking acquisition (as described above). Depending onthe phase shift of the electric fields at the eight electrodes of thefield cage, arbitrary rotational axes can be realized. A rotation of asingle cell around two different axes which run through its center—forexample within the x,y plane of the microscope's field of view andaround the z-axis—enables multiple sets of images to be repeatedlycaptured and the 3-D structure of the cell can be fully evaluated fromall angles, limited only by the number of sampling repeats.

The invention can be combined easily with many different techniques offluorescence microscopy including all confocal techniques as well aswith methods for single molecule detection [e.g. fluorescencecorrelation spectroscopy, Fluorescence Intensity Distribution Analysis(FIDA), Moment Analysis of Fluorescence Intensity Distribution (MAFID)].For example, it can be advantageous to move a light beam that passes theobjective in one dimension like in a laser-scanning microscope(scanning). The object can be rotated slowly around a horizontal (xy)and/or vertical (z) axis, typically with a frequency of 0.5 rps(revolutions per second) to 1 rpm (revolutions per minute). Along thesame lines, a rotation around the z-axis and simultaneous fixed-pointscanning in a single direction (like applied in the “In-sight” reader ofmolecular fluorescence equipped with a linear beam scanner) yields atwo-dimensional scan of a plane within the cell. Photon counting andsignal processing techniques can then count fluorescent molecules withinthis specific plane. Along the same lines, the ability of the inventionto move the suspended cell along the z-axis is of great utility. Inparticular, moving the cell eliminates the need to move the carrier inrelation to the microscope objective. Thus, once the optical system isaligned for multi color observations, chromatic aberration due todifferent refractive behavior of different excitation wavelengths inmulti color applications does not occur.

The Invention Enhances the Application of Existing 3-D RenderingPSF-Based Algorithms.

The ability to rotate a micro object is useful for samplingsub-compartmentalised domains. For example, by grace of the invention, aliving cell may be suspended and imaged in its starting position, beforebeing rotated around an axis within the x,y (equatorial) plane by adefined angle (e.g. 180°, 90° or less) and then imaged again in a secondposition. The same procedure is repeated for an axis perpendicular tothe first in the z plane. This repeating procedure provides a series ofimages of the same cell from different angles which, in principal, canbe used to map the whole 3D volume with a resolution and precision beingdependent from the number of images and different angles included (seebelow). In combination with the ability to rotate the micro-object, theinvention allows small (50 nm-50 μm) z-axis movements to be achieved.Thus, an object may be rotated and shifted on the z-axis in order tobuild a series of images from the same sub-volume, but from differentviews. Clearly, this type of combined rotational/axial manipulationallows objects to be introduced and orientated within the “optimalimaging space” i.e. at a focal plane whereby the application ofPSF-based deconvolution and image reconstruction algorithms may beoptimised according to corrections intended to minimise spatial andchromatic aberration. Such existing methods must always take intoaccount the coordinates of the optical measurement volume, defined bythe position and characteristics of the microscope objective relative toa fixed position sample. When the z-position changes (i.e. the focus ismoved) all these parameters (i.e. those determining the PSF and axialvolume) change and must be recalculated, re-measured or estimated.Inasmuch as the invention allows for the optical geometry to be fixed,it therefore greatly reduces the errors intrinsic to measurement andapplication of existing image algorithms used for deconvolution andvolume reconstruction from z-stack image series.

The Invention Opens the Way for Development of New Three DimensionalImage Reconstruction Algorithms.

As mentioned in the preceding paragraph, in addition to enhancing theapplication of existing algorithms, the invention also opens the way fordevelopment of image processing algorithms previously applied to otherproblems. For example, in X-ray computer tomography objects are imagedby rotation of the detection system around the object. By taking amultiple image series of intermediate coordinates from around theobject, it is possible to both render the object's three-dimensionalvolume, and increase resolution of the images. Thus, by extrapolation,it is in principal possible to apply an analogous algorithmic approachto rendering of micro-object rotational series generated using theinvention described here (e.g. see Appendix I). As such, the inventionunderlies development of completely new “3D Image Reconstruction”methods. The primary advantage of this sort of approach is that theinvention ensures that imaging uses solely the x,y plane of resolutionto visualize a micro object from multiple view points. Given the absenceof axial aberration and reduced z-axis sampling resolution, these newmethods shall implicitly deliver 3D reconstruction representations withsubstantially higher resolution than for conventional reconstructiontechniques. Indeed, such methods may also be combined with those alreadyexisting image algorithms techniques that use a combination of PSF andvolume rendering.

Another subject of the invention is a device for high-resolution imagingof at least one object, which comprises an optical-microscopic imagingsystem with a receptacle for accommodating the object and a controlcircuit for controlling the generation of the intermediate images,wherein this imaging system is equipped with a driving device for movingthe object relative to the receptacle. According to a preferredembodiment of the invention, the receptacle is provided by a fluidicmicrosystem with an arrangement of microelectrodes, wherein the drivingdevice comprises the driving electrodes and a driving circuit within thecontrol circuit.

According to a preferred embodiment of the invention, the controlcircuit contains a switching box being adapted for predeterminedswitching the rotation axis of the object. The switching box allowspredetermined manipulations of the object as well as the implementationof a time trigger.

Further advantageous embodiments of the imaging system are characterizedby a multi-electrode arrangement in the fluidic microsystem. Themulti-electrodes arrangement comprises e.g. at least 3 electrodes withinone plane surrounding the object. These electrodes can be controlled foran improved compensation of position or velocity variations ofasymmetric objects.

Further advantages are derived from the fact that the objectmanipulation in the receptacle does not effect the object (e.g. thephysiology of biological cell). High-resolution imaging according to theinvention can be conducted even over a long period of time (e.g. 60minutes).

ALTERNATIVE EMBODIMENT

The implementation of the invention is not restricted to the applicationwith a microscope. Alternatively, the Method for high-resolution imagerecording of at least one object can be implemented with a measuringdevice comprising a predetermined measurement field. As an example, themeasuring device can comprise an impedance measurement device and saidmeasurement field being the receptacle itself.

4) BRIEF DESCRIPTION OF THE DRAWINGS

Further details and advantages of the invention are described in thefollowing with reference to the attached drawings, which show in:

FIG. 1 a schematic view of an object in a receptacle,

FIG. 2: an arrangement of microelectrodes,

FIG. 3: an illustration of sampling and generation of image-spin seriesaccording to the invention,

FIGS. 4, 5: further illustrations of object movements in a receptacle,

FIG. 6: a schematic view of an imaging system according to theinvention,

FIGS. 7, 8: illustrations of experimental results, and

FIG. 9: an illustration of the elongation effect in conventionalmicroscopes.

5) PREFERRED EMBODIMENTS OF THE INVENTION

Firstly, examples of a method according to the present invention aredescribed with reference to FIGS. 1 to 5. For illustrating the referencedirections, FIG. 1 shows a site view of an object to be investigated(e.g. a biological cell) in a receptacle of an imaging system and theposition of the electrodes at the receptacle, respectively.

FIG. 1 shows the cell 1.4 containing a fluorescent feature 1.5. The cellis caged within the field cage in between the upper glass substrate 1.1carrying the upper electrode plane 1.2 a and the lower glass substrate1.3 carrying the lower electrode plane 1.2 b by means of the electricfield. The cell is rotated around a horizontal axis (1.9) and/or avertical axis (1.10). It is imaged through a microscope objective lens1.11 using light of a specific wavelength 1.7 which is focused to afocal volume element 1.6. x, y and z axes of the optical set-up areindicated in perspective view (1.8).

The position of the electrodes of the dielectric field cage isillustrated in FIG. 2. Reference numerals 1, 2, 3, 4 refer to electrodetips of the four electrodes within the upper electrode plane, i.e. onthe upper glass substrate of the DFC channel.

Reference numerals 1′, 2′, 3′, 4′ refer to electrode tips of the fourelectrodes within the lower electrode plane, i.e. on the lower glasssubstrate of the DFC channel which is facing towards the microscopeobjective. X, y and z are the axes of the optical set-up as described inthe text.

Continuous Sampling

The method of the invention is based on the collection (sampling) of atleast two intermediate images of the object. Sampling can be performedin a continuous fashion as described with reference to FIG. 3. Samplingis achieved by spinning the object for the generation of image-spinseries. The cell is rotated at a constant speed of typically 0.1-60(e.g. approximately 1) rotation per minute and data sets (intermediateimages) are acquired at a speed of typically 1-1000 (e.g. 10) frames persecond.

The cell 3.1, having a dimension 3.2 of several micrometers, iscontinuously sampled during low velocity, high precision rotation of thecell within the z-plane 3.5, i.e. around the y-axis (y). Every feature(e.g. 3.3) of the cell will be within the focal plane 3.7, having athickness of several hundred nanometers, at a certain time and will beimaged there with highest xy resolution. Discontinuous sampling (seebelow), i.e. a xy move of 90° (3.6) followed by a second rotation of thecell within a the z-plane (3.5) allows full sampling. This positions afeature with the start position 3.3 for the first scan to the startposition 3.4.

Discontinuous Sampling

Examples of discontinuous sampling are illustrated in FIG. 4. Thediscontinuous sampling is characterized by a series, or some combinationtherein, of moves comprising rotations and/or z-deflections (4.4) todiscrete points where an aimage is captured, or a rotational image scanis initiated. FIG. 4 shows an example of scanning using multiplerotations. A scan is started at t=0 sec. at position 4.1 with a cellrotation speed of approximately 1 rotation per minute and dataacquisition speed of 10 frames per second. The cell is spun around thespin axis (4.1.1) by the angle (4.1.2). The cell is rotated by 180° toposition 4.2 and 360° to position 4.3. The feature 4.1.3 has thenfulfilled the full circle and the feature 4.1.4 has been sampled twicefrom two distinct positions. The 90° rotation to position 4.4 ispreferably conducted by a switching box being a part of the controlcircuit described below (see FIG. 6)

Piezo-Electric Objective Sampler in Combination with Fixed MultipleSampling Angles

The image-spin series of the invention can be combined to good effectwith fixed and discontinuous sampling in an alternating-mode. FIG. 5shows the procedures using fixed sampling angles of 90°. According toalternative embodiments, other sampling angles can be used.

First, a z-scan is performed on a motionless cell at fixed position 5.1using a piezo-electric objective sampler (schematically shown at 5.1.1).The z-position of the focus is varied in small steps, e.g. 0.1 to 1 μm(z-scan). Reference numeral 5.1.2 indicates an example of an actualfocal plane. After taking a series of images, the cell is rotated aroundthe y-axis 5.1.4 by a fixed angle of 90° (5.1.3) to position 5.2followed by another z-scan (5.2.1). A second series of images is taken.A further rotation by 90° around the x-axis 5.2.2 to position 5.3followed by another z-scan (5.3.1) completes the procedure.

According to the above principle, the feature 5.1.5 is sampled twice intwo perpendicular axes. In position 5.1, x-y-images are taken independence on z:

5.1: (x,y→z)

After turn 5.1.3, the feature 5.1.5 has a new z-position in thex-y-images:

5.1.3, 5.2: (z→x, y)

Both scans 5.1.1 and 5.2.1 deliver a plurality of images, which allow acalculation of the x,y,z-volume of the feature 5.1.5. The feature 5.1.6is not sampled until the second turn. The total object is reconstructedfrom all coordinate stacks after the second rotation around the x-axisonly.

FIG. 6 illustrates a preferred embodiment of a high-resolution imagingdevice according to the invention. The imaging device comprises anoptical microscope imaging system (10), a receptacle (20) and a controlcircuit (30). Preferably, the imaging system (10) comprises aconventional optical microscope (11) equipped with a barrel lens (12)objective, and an imaging camera (13). The camera (13) is e.g. a CCDcamera. Alternatively, photodiodes or multiplier tubes can be used asdetectors. The microscope (11) (shown schematically) can be positionedrelative to the receptacle (20) with a microscope drive (14). The sampleillumination is conducted through the microscope (11) or (shown withbroken lines) with a separate illumination device (15), which can bepositioned on the opposite side of the receptacle (20). The arrangementof FIG. 6 can comprise a laser tweezer device (not shown) foradditionally exerting optical forces to the object.

The receptacle (20) comprises a fluidic microsystem with a channel orchamber (21) for accommodating a liquid with at least one suspendedobject to be investigated. Microelectrodes (22) are arranged on thewalls of the chamber (21) as it is known from conventional microsystemtechnology. The chamber (21) is closed with a cover glass (23) andcarried by a support (24). The support (24) can be adjusted with areceptacle drive unit (25).

The control circuit (30) comprises a data set storage (31), a parameterstorage (32), a switching box (33), a calculation circuit (34) and anobject image storage (35). The control circuit (30) is connected withfurther input/output devices (40), e.g. with a display and a printer.

For conducting the method according to the invention, the object to beinvestigated is positioned in the optical axis of the microscope (11).The microelectrodes are controlled with a certain set of parameters,which are contained in the parameter storage (32). The parameterscomprise e.g. the phase, amplitude and frequency of electrical fields,information about the control mode as well as object properties.According to the parameters, the object is moved within the receptacle(20) as described above. During the movement, a series of at least twodata sets is recorded with the camera (13) and submitted to the data setstorage (31). The data sets are evaluated in the calculation circuit(34) in dependence on the parameters (32) for obtaining the objectimage, which is submitted to the object image storage (35).

The switching box (33) is connected with the parameter storage (32) andthe calculation circuit (34). The switching box (33) is arranged forsubmitting predetermined switching parameters to the parameter storage(32). According to the switching parameters, a movement mode of theobject in the receptacle (20) is adjusted.

EXAMPLES Imaging Experiments Performed with a Prototypic Set-Up Based onthe Invention

—A—Visualization of the Lamin Structure of HeLa Cells

As an example for studying a cellular protein by live three-dimensionalmicroscopy fluorescent nuclear lamin proteins inside a live suspendedcell were imaged using a prototype of the invention.

Lamins are the major components of the nuclear lamina, a two-dimensionalfilamentous network at the periphery of the nucleus in highereukaryotes, directly underlying the inner nuclear membrane. In thecourse of cell-cycle-dependent dynamics of the nucleus in highereukaryotes, lamins as well as lamin-binding proteins seem to possessimportant functions during various steps of post-mitotic nuclearreassembly and seem to play a role in various pathological processes inmuscle (Emery-Dreifuss muscular dystrophy, EDMD; dilated cardiomyopathyand conduction system defect, DCM-CD; and limb-girdle musculardystrophy, LGMD) and adipose tissue (Dunnigan-type familial partiallipodystrophy, FPLD). Lamins are more dynamic than originally thought.These findings altogether demonstrate the necessity to investigate thelocalization of lamins within the cell and their dynamics with greatprecision in order to learn about their functions and dysfunctions. HeLacells were used for this purpose which overexpressed lamin protein fusedto the fluorescent protein dsRed.

The cells were detached from the culture flask using trypsin andsuspended in Cytocon™ Buffer II containing 30 mM salt/phosphate bufferbalanced to normo-osmolar values using 0.3 M inositol solution. Thecells were introduced into DFC4 chips and caged within the 30 μm fieldcage (distance between opposing electrode tips within one plane: 30 μm)using the Cytocon™ 300. The field cage was energized at 700 kHz and1.9-2.6 Vrms. The cells were subjected to a rotational movement by usingthe “rot II” mode of the Cytocon™ 300. The DFC4 Chips was mounted on aZeiss Axiovert 200 microscope equipped with the Photometrics Cool SNAPcamera Hamamatsu and the T.I.L.L. Vision imaging system. The cellrotated at a constant speed of approximately 20 revolutions per minuteand an image-spin series was generated at a speed of 10 frames persecond with 50 ms exposure time. The axis of the rotation was selectedby a controlled modulation of amplitudes of the upper and lowerelectrode plane of the field cage.

FIG. 7 shows a diagram of fluorescence intensity within an image area ofthe HeLa cell during the image-spin series of the experiment describedhere and partially shown below in FIG. 9. The fluorescence intensity inarbitrary units is plotted against the number of the images taken at arate of 10 per second. The position of the peaks demonstrate theaccuracy of the frequency which can be achieved for a cell spin. Theperiod can be determined from the distance of two peaks, e.g., betweenpeak 7-1 and 7-2.

Image-spin series of the HeLa cell with overexpressed lamin proteinfused to the fluorescent protein dsRed are illustrated in FIG. 8. FIG.8-1 show the image taken at second 4, FIG. 8-2 is the image taken atsecond 21 and FIG. 8-3 is the image taken at second 34. The fluorescentobject 8-4 occurs in image 8-1 at a specific position given by axy-coordinate 8-5 and it appears at an identical position in the image8-3 after ten revolutions.

—B—Rotation of Jurkat Cells Around Two Different Rotation Axes

Jurkat-cells were suspended in Cytocon™ Buffer II and introduced into aDFC Chip. A single cell was caged within the center 30 μm field cage ofthe chip using a Cytoman™ and a frequency of 700 kHz and an amplitude of1.9-2.6 Vrms. The cell could be continuously rotated around a horizontalas well as a vertical axis without altering its center position. Spinfrequency could be varied from 0.01 rps (revolutions per second) to 0.5rps by altering the amplitude in a range of 1.5-3 Vrms and/or thefrequency in a range of 0.3 MHz-8 MHz.

The Cytoman™ provides two different rotational field modes and onealternating field mode according to table 1. A switch box enabled tochange between two different spin axes (around a vertical and ahorizontal spin axis, see FIG. 2).

TABLE 1 Addressing of the eight DFC electrodes for a cell spin around avertical and a horizontal axis as well as for motionless positioning (norotation). The phase shift of the field vector is given (Schnelle, Th.et al. “J. Electrostatics” 50, 2000, 17-29). Electrodes 1 2 3 4 1′ 2′ 3′4′ Vertical Rotational 0 π/2 π −π/2 π −π/2 0 π/2 spin axis field I (z)Horizontal Rotational 0 π/2 −π/2 π −π/2 π 0 π/2 spin axis field II (x,y) Fixed Alternating 0 π 0 π 0 π 0 π position field, type C

APPENDIX I

A deconvolution, volume reconstruction operation bases itself on thefollowing considerations: “A digital image (e.g. microscopic image of adistribution of fluorescent molecules) is a convoluted 2-dimensionalrepresentation of a 3-dimensional reality. The individual informationgiven at each image pixel includes not only the true signal emergingfrom a single point in the focal plane, but also varying degrees ofadditional signal accumulated from fluorescent out-off-focus volumes,above and below the focal plane, but within the focal depth alongz-axis” (from Shorte & Bolsover, 1999). Fluorescent specimens act asself-luminous objects in which point sources behave as independentsources. Neglecting the light scattering, the optical density g at eachpixel can be written as:g=∫Hcdzwherein H is the extinction coefficient and c the absorberconcentration. For reconstructing a 3-dimensional image of the object, cis to be obtained from entities g and H for a plurality of image planesby deconvolution.

The deconvolution is obtained in analogy to conventional reconstructionfrom radiographic projections (tomography). In standard tomography, thespecimen does not move but the radiographic apparatus turns around it toget a set of images. The values c can be reconstructed as described byB. Chalmond et al. in “inverse problems”, vol. 15, 1999, p. 399-411.According to the present invention, the situation is reversed. Theobject to be investigated is rotated whereas the microscope is fixed.The conventional tomography algorithms can be used after a numericalcoordinate transformation of the microscope set up into the radiographiccontext.

1. A method for high-resolution image recording of at least one objectwith a microscope, comprising the steps of: positioning the at least oneobject in a receptacle arranged in an optical axis of the microscope,generating at least two first data sets per object, wherein: (a) the atleast two first data sets represent intermediate images of the at leastone object with at least two different predetermined orientationsrelative to the optical axis of the microscope, (b) the at least twodifferent predetermined orientations of the object are provided bycontrolled movement of the at least one object relative to thereceptacle, and (c) the controlled movement comprises a rotation of theat least one object by an influence of electrical field forces, saidobject being rotated around at least one of a predetermined axis and apredetermined rotation angle, and evaluating the data sets to obtainquantitative three-dimensional information of the at least one object,wherein said quantitative three-dimensional information represents athree-dimensional shape of the at least one object.
 2. The methodaccording to claim 1, wherein said moving of the at least one objectrelative to the receptacle further comprises a translation of the atleast one object by the influence of the electric field forces.
 3. Themethod according to claim 2, wherein said rotation comprises at leastone rotation with a rotation axis parallel to the optical axis.
 4. Themethod according to claim 2, wherein said rotation comprises at leastone rotation with a rotation axis slanted relative to the optical axis.5. The method according to claim 4, wherein said rotation axis isslanted within an angle range of up to 90°.
 6. The method according toclaim 2, wherein said rotation comprises: a rotation in a continuousmode or for predetermined time periods and angles, and/or a rotationwith changing rotational axes.
 7. The method according to claim 2,wherein said rotation is conducted by holding the at least one object ata fixed position by said electric field forces and by rotating the atleast one object by optical forces.
 8. The method according to claim 1,further comprising steps of generating further intermediate images ofthe object, each with another focal plane, respectively, wherein eachsaid focal plane is adjusted by scanning an objective of the microscopeparallel to the optical axis.
 9. The method according to claim 8,wherein said at least two different predetermined orientations of theobject and said scanning an objective are conducted in an alternatingmode.
 10. The method according to claim 1, wherein said positioningcomprises suspending said at least one object in a liquid in saidreceptacle.
 11. The method according to claim 1, wherein said step ofevaluating the data sets comprises at least one step selected from thegroup consisting of removing out-of-focus light and reconstructing athree-dimensional map/image of the at least one object.
 12. The methodaccording to claim 1, wherein said at least one object comprises atleast one eukaryotic cell, at least one prokaryotic cell and/or at leastone artificial particle.
 13. The method according to claim 1, whereinsaid microscope is used as a fluorescence microscope, a phase contrastmicroscope, a differential interference contrast microscope or aconfocal microscope.
 14. An imaging device for high-resolution imagerecording of at least one object, comprising: a microscope imagingsystem with an optical axis, a receptacle for accommodating said atleast one object, said receptacle being arranged in said optical axis,and a control circuit being arranged for: (a) generating at least twofirst data sets per object, wherein said at least two first data setsrepresent intermediate images of the at least one object with at leasttwo different predetermined orientations relative to the optical axis,(b) providing controlled movement of the at least one object relative tothe receptacle to generate the at least two different predeterminedorientations, wherein the controlled movement comprises a rotation ofthe at least one object by an influence of electrical field forces, saidobject being rotated around at least one of a predetermined axis and apredetermined rotation angle, and (c) evaluating the data sets to obtainan object image representing a three-dimensional shape of the at leastone object, and a driving device adapted to controllably move the atleast one object relative to the receptacle.
 15. The imaging deviceaccording to claim 14, wherein said receptacle comprises a chamber of afluidic microsystem and said driving device comprises microelectrodesarranged at walls of said chamber and connected with said controlcircuit.
 16. The imaging device according to claim 15, wherein saiddriving device comprises at least three microelectrodes arranged in oneplane in said chamber.
 17. The imaging device according to claim 16,wherein said driving device comprises at least six microelectrodesarranged in two planes in said chamber.
 18. The imaging device accordingto claim 14, wherein said control circuit comprises a switching boxarranged for switching a rotation axis of the at least one object.
 19. Amethod for high-resolution image recording of at least one object with ameasuring device with a predetermined measurement field, comprising thesteps of: positioning the at least one object in a receptacle arrangedin the measurement field of the measuring device, generating at leasttwo first data sets per object, wherein: (a) the at least two first datasets represent intermediate data of the at least one object with atleast two different predetermined orientations relative to themeasurement field of the measuring device, (b) the at least twodifferent predetermined orientations of the object are provided bycontrolled movement of the at least one object relative to thereceptacle, and (c) the controlled movement comprises a rotation of theat least one object by an influence of electrical field forces, saidobject being rotated around at least one of a predetermined axis and apredetermined rotation angle, and evaluating the data sets to obtainquantitative three-dimensional information of the at least one object,wherein said quantitative three-dimensional information represents thethree-dimensional shape of the at least one object.
 20. The methodaccording to claim 19, wherein said measuring device comprises amicroscope and said measurement field is an optical axis of themicroscope.
 21. The method according to claim 19, wherein said measuringdevice comprises an impedance measurement device and said measurementfield is the receptacle.